The role of the free energy landscape in Parkin's function and dysfunction in health and disease

自由能景观在健康和疾病中帕金功能和功能障碍中的作用

基本信息

批准号：
9883915
负责人：
A. JOSHUA WAND
金额：
$ 32.69万
依托单位：
TEXAS A&M AGRILIFE RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-01 至 2024-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9883915
关键词：
Address Allosteric Regulation Binding Binding Proteins Biological Biology Calorimetry Cardiac Cardiomyopathies Catalytic Domain Characteristics Clinic Clinical Competence Complex Coupling Data Defect Disease Dissection Elements Entropy Enzymatic Biochemistry Enzymes Eukaryota Exons Fluorescence Foundations Free Energy Functional disorder Goals Health Heart Diseases Hydration status Hydrogen Intervention Knowledge Lead Link Malignant Neoplasms Mass Spectrum Analysis Measures Methods Micelles Mitochondria Modernization Molecular Conformation Monitor Mutagenesis Mutation Myocardium NMR Spectroscopy Neurologic Neurons PINK1 gene Parkin gene Parkinson Disease Pathologic Phosphotransferases Point Mutation Post-Translational Protein Processing Process Proteins Proxy Regulation Regulatory Element Relaxation Relaxation Techniques Repression Role Signal Transduction Site Structure Structure-Activity Relationship Tertiary Protein Structure Testing Thermodynamics Ubiquitin Water base biophysical techniques disease-causing mutation early onset human disease insight member mitochondrial autophagy molecular recognition mutant nervous system disorder novel parkin gene/protein proteostasis single molecule small molecule ubiquitin ligase ubiquitin-protein ligase

项目摘要

The RING ubiquitin E3 ligases are a superfamily of proteins critical to protein homeostasis and signaling in eukaryotes. Dysfunctions in E3 ligases are implicated in innumerable human diseases. This proposal focuses on the regulation of the ubiquitin E3 ligase Parkin. Parkin is central to the controlled destruction of damaged mitochondria by autophagy (mitophagy). Controlled mitophagy is particularly essential to cardiac and neuronal health. Uncontrolled mitophagy due to mutations in Parkin is clearly a driver of early onset Parkinson's disease (eoPD). Parkin is now implicated in a number of other neurological diseases, cardiomyopathy and in various cancers. The central goal here is to create an understanding the physical basis for regulation of Parkin and how clinically observed mutations promote unregulated activity leading to inadequately controlled mitophagy and other biological defects. Though much is known about the biology and structural basis of Parkin function, very little is certain about the physical basis for its regulation. Parkin activity is suppressed by its intra-molecular association with a ubiquitin-like domain and is allosterically activated by the binding of phosphorylated ubiquitin (pUb). Phosphorlyation of the Ubl domain also promotes activation. This complicated intersection of regulatory mechanisms can only be understood by the rigorous dissection of the underlying thermodynamics. Without this knowledge one cannot fully interpret the effects of mutations that lead to disease. We shall take advantage of the broad foundation of knowledge of the biology of Parkin and structural basis of its function to address the poorly understood thermodynamics of allosteric regulation of Parkin. The basis for regulatory control of Parkin will be cast in a modern statistical thermodynamics description of the protein ensemble. The influence of allosteric regulators and post-translational modifications will be examined by comprehensive hydrogen exchange monitored by mass spectrometry and NMR spectroscopy; advanced NMR relaxation techniques; single molecule fluorescence; calorimetry; enzymology; and mutagenesis. A more rigorous and complete understanding of the regulation of Parkin will enable a robust interpretation of pathological mutations. Not all pathological mutations can be simply explained as mutations that disrupt the levels of protein or mutations that directly impact the catalytic site. Examples of common pathological mutations will be examined to reveal the basis for their effects on Parkin's regulatory fidelity, with a longer- range goal of determining how this impact might be mitigated by small molecule intervention.

环泛素E3连接酶是对蛋白质稳态至关重要的蛋白质的超家族，在真核生物。 E3连接酶中的功能障碍与无数的人类疾病有关。该提案重点是关于泛素E3连接酶Parkin的调节。帕金是受控破坏的核心通过自噬（线粒体）损坏的线粒体。受控线粒体对心脏尤其重要和神经元健康。由于帕金突变引起的不受控制的线粒体显然是早期发作的驱动力帕金森氏病（EOPD）。帕金现在与其他许多神经系统疾病有关，心肌病和各种癌症。这里的核心目标是建立理解物理调节Parkin的基础以及临床观察到的突变如何促进不受管制的活性导致无法充分控制的线粒体和其他生物缺陷。尽管对帕金功能的生物学和结构基础知之甚少，但对其监管的物理基础。帕金蛋白的活性被其与A的分子内关联所抑制泛素样结构域，并通过磷酸化的泛素（PUB）的结合而被短构激活。 UBL结构域的磷光化也促进了激活。这个复杂的调节交集只有通过严格的基础热力学的严格解剖才能理解机制。没有这种知识不能完全解释导致疾病的突变的影响。我们将利用帕金生物学和结构基础知识的广泛基础它的功能是解决帕金变构调节的热力学知识。基础对于帕金的监管控制，将以现代的统计热力学描述进行蛋白质合奏。变构调节器和翻译后修改的影响将是通过质谱和NMR光谱法监测的综合氢交换检查；先进的NMR松弛技术；单分子荧光；量热；酶学；和诱变。对Parkin的调节的更严格，更完整的理解将实现强大的解释病理突变。并非所有病理突变都可以简单地解释为破坏的突变直接影响催化位点的蛋白质或突变的水平。常见病理的例子将检查突变，以揭示其对帕金监管保真度的影响的基础，并具有更长的时间小分子干预可能如何减轻这种影响的范围目标。